1. Field of the Invention
The present invention relates to novel calcium phosphate core particles, to methods of making them, and to methods of using them as vaccine adjuvants, as cores or carriers for biologically active material, and as controlled release matrices for biologically active material.
2. Description of Related Art
Nanometer scale particles have been proposed for use as carrier particles, as supports for biologically active molecules, such as proteins, and as decoy viruses. See U.S. Pat. Nos. 5,178,882; 5,219,577; 5,306,508; 5,334,394; 5,460,830; 5,460,831; 5,462,750; and 5,464,634, the entire contents of each of which are hereby incorporated by reference.
The particles disclosed in the above-referenced patents, however, are generally extremely small, in the 10-200 nm size range. Particles of this size are difficult to make with any degree of consistency, and their morphology is not described in any detail. None of these patents disclose the use of nanoparticles as sustained release matrices. Furthermore, these patents do not disclose the use of calcium phosphate particles as either (1) adjuvants for vaccines or viral decoys, or (2) controlled release matrices for delivery of pharmaceuticals or immunogenic materials.
There has been a suggestion in the literature to use calcium phosphate particles as vaccine adjuvants, but calcium phosphate particles have generally been considered an unsuitable alternative to other adjuvants due to inferior adjuvanting activity. See, e.g., Goto et al., Vaccine, vol. 15, no. 12/13 (1997). Moreover, the calcium phosphate evaluated was typically microparticulate ( greater than 1000 nm diameter) and possessed a rough and oblong morphology, in contrast to the core particles of the present invention.
Therefore, an important need remains for calcium phosphate core particles useful as core materials or carriers for biologically active moieties which can be produced simply and consistently. A further need remains for calcium phosphate core particles that can be effectively used as adjuvants for vaccines, as cores or carriers for biologically active molecules, and as controlled release matrices.
There is also a need for calcium phosphate core particles that can be effectively used as supports and matrices for sustained release of polynucelotide material (DNA or RNA) encoding immunogenic polypeptides. Traditional vaccination involves exposing a potential host to attenuated or killed pathogens, or immunogenic components thereof (e.g., proteins or glycoproteins). The basic strategy has changed little since the development of the first smallpox vaccine nearly a century ago, although modern developments permit genetic engineering of recombinant protein vaccines. However, traditional vaccine methodologies may be undesirable as a result of their expense, instability, poor immunogenicity, limited heterogeneity and potential infectivity.
Polynucleotide vaccination presents a different vaccine methodology, whereby polynucelotide material, such as DNA or RNA, encoding an immunogenic polypeptide is delivered intracellularly to a potential host. The genetic material is taken up and expressed by these cells, leading to both a humoral and a cell-mediated immune response. It is not entirely clear whether DNA vaccines function as a result of integration or simply long-term episomal maintenance.
Polynucleotide vaccination provides numerous advantages over traditional vaccination. Polynucleotide vaccines eliminate the risk of infection associated with live attenuated viruses, yet advantageously induce both humoral and cell-mediated responses. Polynucleotide vaccines further provide prolonged immunogen expression, generating significant immunological memory and eliminating the need for multiple inoculations. Polynucleotide vaccines are very stable, permitting prolonged storage, transport and distribution under variable conditions. As a further advantage, a single polynucleotide vaccine may be engineered to provide multiple immunogenic polypeptides. Thus, a single DNA vaccine can be used to immunize against multiple pathogens, or multiple strains of the same pathogen. Finally, polynucleotide vaccines are much simpler and less expensive to manufacture than traditional vaccines.
Polynucleotide vaccines may take various forms. The genetic material can be provided, for example, in combination with adjuvants capable of stimulating the immune response. Administration of the DNA or RNA coated onto microscopic beads has been suggested. See J. J. Donnelly et al., Annu. Rev. Immunol. 15, 617 (1997). Various routes of administration are also possible, and may include, for example, intravenous, subcutaneous and intramuscular administration.
A desirable immune response to an immunogenic polypeptide is two-fold, involving both humoral and cellular-mediated immunity. The humoral component involves stimulation of B cells to product antibodies capable of recognizing extracellular pathogens, while the cell-mediated component involves T lymphocytes capable of recognizing intracellular pathogens. Cytotoxic T-lymphocytes (CTLs) play an important role in the latter, by lysing virally-infected or bacterially-infected cells. Specifically, CTLs possess receptors capable of recognizing foreign peptides associated with MHC class I and/or class II molecules. These peptides can be derived from endogenously synthesized foreign proteins, regardless of the protein""s location or function within the pathogen. Thus, CTLs can recognize epitopes derived from conserved internal viral proteins (J. W. Yewdell et al., Proc. Natl. Acad. Sci. (USA) 82, 1785 (1985); A. R. M. Towsend, et al., Cell 44, 959 (1986); A. J., McMichael et al., J. Gen. Virol. 67, 719 (1986); A. R. M. Towsend and H., Annu. Rev. Immunol. 7, 601 (1989)) and may therefore permit heterologous protection against viruses with multiple serotypes or high mutation rates. Polynucleotide vaccination can stimulate both forms of immune response, and thus is very desirable.
Efforts to use polynucleotide vaccination have focused on the use of viral vectors to deliver polynucleotides to host cells. J. R. Bennink et al., 311, 578 (1984); J. R. Bennink and J. W. Yewdell, Curr. Top. Microhiol. Immunol. 163, 153 (1990); C. K. Stover et al., Nature 351, 456 (1991); A. Aldovini and R. A. Young, Nature 351, 479 (1991); R. Schafer et al., J. Immunol. 149, 53 (1992); C. S. Hahn et al., Proc. Natl. Acad. Sci. (USA) 89, 2679 (1992). However, this approach may be undesirable for several reasons. Retroviral vectors, for example, have restrictions on the size and structure of polypeptides that can be expressed as fusion proteins while maintaining the ability of the recombinant virus to replicate (A.D. Miller, Curr. Top. Microbiol. Immunol. 158, 1 (1992). The effectiveness of vectors such as vaccinia for subsequent immunizations may be compromised by immune responses against vaccinia (E. L. Cooney et al., Lancet 337, 567 (1991)). Also, viral vectors and modified pathogens have inherent risks that may hinder their use in humans (R. R. Redfield et al., New Engl. J Med. 316, 673 (1987); L. Mascola et al., Arch. Intern. Med. 149, 1569 (1989)). For example, in live vector approaches, highly immunogenic vectors also tend to be highly pathogenic.
Alternative gene delivery methods have also been explored. Benvenisty, N., and Reshef, L. (PNAS 83, 9551-9555, (1986)) showed that CaCl2 precipitated DNA could be expressed in mice. Plasmid vectors have also been used to produce expression in mouse muscle cells (J. A. Wolff et al., Science 247, 1465 (1990); G. Ascadi et al., Nature 352, 815 (1991)). The plasmids were shown to be maintained episomally and did not replicate. Subsequently, persistent expression has been observed after i.m. injection in skeletal muscle of rats, fish and primates, and cardiac muscle of rats (H. Lin et al., Circulation 82, 2217 (1990); R. N. Kitsis et al., Proc. Nafl. Acad. Sci. (USA) 88, 4138 (1991); E. Hansen et al., FEBS Lett. 290, 73 (1991); S. Jiao et al., Hum. Gene Therapy 3, 21 (1992); J. A. Wolff et al., Human Mol. Genet. 1, 363 (1992)). WO 90/11092 (Oct. 4, 1990) reported the use of naked polynucleotides to vaccinate vertebrates.
Various routes of administration have been found to be suitable for vaccination using polynucleotide vaccines. Intramuscular administration is thought to be particularly desirable, given the proportionally large muscle mass and its direct accessibility through the skin. See U.S. Pat. No. 5,580,859. Tang et al., (Nature, 356, 152-154 (1992)) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of anti-BGH antibodies in the mice. Furth et al., (Analytical Biochemistry, 205, 365-368, (1992)) showed that a jet injector could be used to transfect skin, muscle, fat, and mammary tissues of living animals. WO 93/17706 describes a vaccination method wherein carrier particles are coated with a gene construct and then accelerated into a potential host. Intravenous injection of a DNA:cationic liposome complex in mice has also been reported (Zhu et al., Science 261, 209-211 (Jul. 9, 1993); see also WO 93/24640). Methods for introducing nucleic acids have been reviewed (Friedman, T., Science, 244, 1275-1281 (1989)), see also Robinson et al., (Abstracts of Papers Presented at the 1992 meeting on Modern Approaches to New Vaccines, Including Prevention of AIDS, Cold Spring Harbor, p 92; Vaccine 11, 957 (1993)), where the intra-muscular, intra-venous, and intra-peritoneal administration of avian influenza DNA into chickens was alleged to have provided protection against lethal challenge.
Reports suggest that polynucleotide vaccination has provided effective protective immunity in various animal models. The immunization of mice against influenza by the injection of plasmids encoding influenza A hemagglutinin has been reported (Montgomery, D. L. et al., 1993, Cell Biol., 12, pp. 777-783), or nucleoprotein (Montgomery, D. L. et al., supra; Ulmer, J. B. et al., 1993, Science, 259, pp. 1745-1749). The first use of DNA immunization for a herpes virus has been reported (Cox et al., 1993, J. Virol., 67, pp. 5664-5667). Injection of a plasmid encoding bovine herpes virus 1 (BHV-1) glycoprotein g IV gave rise to anti-g IV antibodies in mice and calves. Upon intranasal challenge with BHV-1, immunized calves showed reduced symptoms and shed substantially less virus than controls. Wang et al., (P.N.A.S. USA 90, 4156-4160 (May, 1993)) reported on elicitation of immune responses in mice against HIV by intramuscular inoculation with a cloned, genomic (unspliced) HIV gene. However, the level of immune responses achieved was very low, and the system utilized portions of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter and portions of the simian virus 40 (SV40) promoter and terminator. SV40 is known to transform cells, possibly through integration into host cellular DNA. Thus, the system described by Wang et al., may be inappropriate for administration to humans.
It has been suggested to use calcium phosphate particles as agents for transfection of therapeutic polynucleotides in gene therapy. See U.S. Pat. No. 5,460,831. DNA or RNA is attached to the particulate core and delivered to a target cell, resulting in expression of therapeutic proteins. However, this patent does not suggest the use of calcium phosphate particles as supports for DNA or RNA vaccines. To the contrary, this patent indicates that the stimulation of an immunological response during transfection is to be avoided. This patent also fails to suggest the use of calcium phosphate particles as controlled release matrices for genetic material.
There is also a need for calcium phosphate core particles that can be used effectively used as an inhalable aerosol delivery system for the delivery of therapeutic proteins or peptide agents, and in particular, for delivery of insulin and other hormones. For a number of therapeutic agents, delivery of the agent to a patient in need thereof can be difficult. This is particularly true with proteins and peptides, which are difficult or impossible to administer orally, since they are easily digested or hydrolyzed by the enzymes and other components of gastric juices and other fluids secreted by the digestive tract. Injection is often the primary alternative administration method, but is unpleasant, expensive and is not well tolerated by patients requiring treatment for chronic illnesses. In particular, patients who are administered drugs on an out-patient basis, or who self-administer, are more likely to fail to comply with the required administration schedule. A particular group of patients of this type are those suffering from diabetes, who frequently must inject themselves with insulin in order to maintain appropriate blood glucose levels.
Recently, alternative methods of administration therapeutic agents have been sought, in particular, administration by inhalation of an aerosol containing the therapeutic agent. The lungs can be used effectively to get the therapeutic agent into the bloodstream because they have a very large surface area of very thin tissue. As a result, for some therapeutic agents and delivery systems, the level of agent in the blood can rise as fast as, or faster than, that obtained when the agent is administered by injection beneath the skin. Moreover, the thin lung tissue allows the passage of proteins and peptides into the blood stream without exposing them to the type or level of proteases encountered during oral administration.
Aerosols containing the therapeutic agent as fine, suspended mists of particles in both liquid and solid form have been investigated. However, preparation of suitable inhalable aerosols can be difficult for therapeutic agents where the blood level of the agent is critical, e.g., with insulin, because the amount of aerosol delivered to the deep lung tissue can be substantially variable, leading to inconsistent dosages of the drug to the patient.
As a result of this need to provide a reliable inhalable aerosol delivery system, various attempts have been made to develop small, solid particles for the delivery of therapeutic agents via inhalation. For example, an inhalable form of insulin is reportedly under development wherein the insulin is combined with sugar particles of a particular size to make an ultrafine powder that is delivered when it is forced through an inhaler nozzle by a blast of compressed air. See R. F. Service, Science 277:5330 (1997). Another inhalable form of insulin involves relatively large (diameters  greater than 5 xcexcm), porous polymer particles (50:50 poly(lactic acid-co-glycolic acid) of low density (xcfx81 less than xcx9c0.4 g/cm3) that encapsulate insulin. The particles are believed to penetrate deep into the lung tissue as the result of their low density, yet avoid phagocytosis when in the tissue as the result of their large size. See D. E. Edwards et al., Science, 276:1868 (1997).
Despite these attempts, there remains a need for an inhalable aerosol delivery system that effectively provides consistent, reliable, therapeutic blood levels of protein or peptide therapeutic agents, and in particular, of insulin and other hormones. It is particularly desirable that any carrier material be very small and easily biodegradable, in order to avoid complications resulting from inhalation of particulates.
The present invention relates to novel calcium phosphate (xe2x80x9cCAPxe2x80x9d) core particles, to methods of making them, and to methods of using them as vaccine adjuvants, as cores or carriers for biologically active material, and as controlled release matrices for biologically active material. More particularly, the invention relates to the core particles having a diameter between about 300 nm and about 4000 nm, more particularly between about 300 nm and about 1000 nm, and having a substantially spherical shape and a substantially smooth surface.
The present invention also relates to the novel calcium phosphate core particles having a material coated on the surface of the core particles, and/or dispersed or impregnated within the core particles, to methods of making them, and to methods of using them. Non-limiting examples of a suitable material to be at least partially coated on the surface of the core particle or impregnated therein include one or more of the following: antigenic material, natural immunoenhancing factors, polynucleotide material encoding immunogenic polypeptides, or therapeutic proteins or peptides.
The present invention also relates to combinations of this novel core particle having at least a partial coating of a surface modifying agent or a surface modifying agent impregnated therein or both. If one or more of the above-mentioned materials (e.g., antigenic material, natural immunoenhancing factors, polynucleotide material, or therapeutic proteins or peptides) is at least partially coated on the particle, the material may be optionally attached to the particle by the surface modifying agent, which acts as a biological xe2x80x98glue,xe2x80x99 such as cellobiose or polyethylene glycol (PEG).
The invention also relates to combinations of this novel core particle with antigenic material, natural immunoenhancing factors, polynucleotide material, or therapeutic proteins or peptides integrated into the core particle, forming a controlled release matrix that releases the material into a patient over time.
One embodiment of the present invention relates to methods of adjuvanting vaccines, whether live, killed, attenuated, a decoy virus, or made from core particles at least partially coated with microbial antigenic material, or combinations thereof, by administering the novel uncoated core particles or core particles coated with natural immunoenhancing factor to a patient in need of vaccination either alone or in combination or conjunction with administration of the vaccine. The core particles are sufficiently small to be easily transportable to various tissues throughout the body, and are biodegradable as well.
The invention also relates to methods of vaccinating patients in need thereof by administering the novel core particle in combination or in conjunction with an antigenic material or natural immunoenhancing factor, wherein the antigenic material or natural immunoenhancing factor is at least partially coated on the core particle and/or integrated therein, as described in more detail below. The calcium phosphate core particles of this embodiment significantly increase the efficacy of the vaccines with which they are administered, by enhancing the magnitudes, qualities, and/or durations of the immune responses.
In another embodiment, the invention also relates to a polynucleotide vaccine having polynucleotide material at least partially coated on the novel core particle and/or impregnated therein. Contrary to conventional wisdom, the present inventors have discovered novel calcium phosphate particles that can be effectively used as supports and matrices for sustained release of DNA or RNA encoding immunogenic polypeptides. The present inventors have discovered that a DNA or RNA vaccine can be prepared that uses a biodegradable matrix of calcium phosphate, that functions as a sustained release composition, conferring long lasting immunity, and that is, in effect, self-adjuvanting. The primary intent is that the respective protein translation products produced by the present invention would immediately be available both intracellularly and extracellularly, to elicit enhanced humoral and cellular immune responses.
When administered as a polynucleotide vaccine, the calcium phosphate in the core particles of the present invention biodegrades, releasing into the surrounding tissue polynucelotide material (DNA or RNA) coding for immunogenic polypeptides. Without wishing to be bound to any theory, it is believed that cells in the patient take up the DNA or RNA and express it as immunogenic proteins, which are then presented to B cells and T cells of the immune system, resulting in both a humoral and cell-mediated response similar to that obtained using live attenuated virus, but without the risks of pathogenicity and without the loss of immunogenicity associated with live virus. When the DNA or RNA is impregnated or dispersed within the calcium phosphate core particle, the gradual release of genetic material by the dissolution of the calcium phosphate matrix provides longer lasting immune responses than does administration of a conventional DNA or RNA vaccine.
In addition, while not wishing to be bound by any theory, it is believed that the presence of calcium phosphate core particles enhances the immune response to the antigenic proteins produced by the cells that take up and express the DNA or RNA, further multiplying the protective effect of the vaccine. The size of the core particles of the invention allows them to migrate through the body as the calcium phosphate gradually degrades, thereby transporting the DNA/RNA to different tissues in the body, and enlisting large numbers of different tissues at different locations in the production of antigenic proteins.
In still a further embodiment, this invention relates to an inhalable, aerosolizable therapeutic composition, having a therapeutic protein or peptide material either at least partially coated on the novel calcium phosphate core particle and/or impregnated therein. The surface of the core particle may be at least partially coated with a surface modifying agent that bonds proteins or peptides to the core particle without denaturing the proteins or peptides. A therapeutic protein or peptide, in particular a hormone such as insulin, is disposed on the resulting coated core particle.
The present invention also relates to methods of treating medical conditions resulting from protein or peptide deficiencies by administering effective amounts of the core particles of this particular embodiment to a patient in need thereof via inhalation into the lungs. The therapeutic compositions of the present invention are highly stable, and exhibit enhanced bioavailability. These therapeutic compositions also exhibit preferable biodynamics including controlled release of therapeutic polypeptides or proteins.
The present invention also relates to methods of preparing the novel calcium phosphate core particles described above, such as the core particles for use individually, the core particles having material at least partially coated on the surface, and the core particles having material impregnated therein.
The above discussed and many other features and attendant advantages of the present invention are detailed below.